SUMMARY CULLIN-RING E3 ubiquitin ligases (CRLs) constitute the largest class of E3s and control a large fraction of regulated proteolysis in human cells. CRLs employ ~200 adaptor proteins to bind and ubiquitylate substrates, and their dynamic assembly with adaptor proteins is regulated by the neddylation cycle together with the CAND adaptor exchange factor. Several CRLs have been linked with turnover of proteins involved in aging, cell cycle control, DNA repair, transcription, and cellular signaling pathways. Despite these advances, our understanding of the CRL system is incomplete for several reasons. First, most studies have focused on a small number of well studied adaptors and a large fraction of adaptors have not been examined, leaving major gaps in our understanding of the many roles these proteins may play. Second, the majority of studies have examined processes in cancer cell lines in culture and we know very little about the physiological roles of CRL adaptors in cell fate decisions such as differentiation. Third, although we know that CRL-adaptor pairs are under dynamic control, we have only a rudimentary understanding of the rules that control adaptor choice during changes in cell state. Fourth, a major challenge for the field has been matching individual adaptors to substrates. Many adaptors have multiple substrates and understanding the rules that control substrate selection has important implications for the field. Here, we propose a series of experiments that seek to broadly address these limitations in our understanding of CRL function and biology. In one series of experiments (AIM 1), we will build on preliminary data to elucidate the roles of CRLs, their adaptors, and the co-E3 ARIH1 in the differentiation of human embryonic stem cells (hESCs) to the three germ cell lineages. This includes genetic experiments that identify critical adaptors in the differentiation process, quantitative proteomic analysis of CRL complex and total proteome remodeling during differentiation, and elucidation of substrates and regulatory mechanisms for individual CRLs adaptors linked with formation of germ layer lineages. In a second series of experiments (AIM 2), we will capitalize on the development of new Global Protein Stability ORF and peptidome libraries to systematically discover targets and degrons on CRLs on a global scale. GPS-Peptidome libraries represent the entire human proteome tiled as 90-mers and provides a means by which to screen for linear degrons which often serve as interaction sites for ubiquitin ligases. In preliminary studies, we have discovered a novel mechanism for recognition of proteins for ubiquitylation involving C-terminal sequences enriched in Gly. This new pathway, which we refer to as the C-end rule pathway, is controlled by a small set of CUL2- based CRLs, and the proposed experiments will further elucidate the components and mechanisms regulating this new pathway which has the potential to regulate a substantial fraction of the proteome. Together, these studies will quantitatively and globally address major gaps in our understanding of physiological functions of CRLs and mechanisms by which substrates are selected for ubiquitylation.